This competing renewal continues to be based on the need for design and development of new biomaterials that will be utilized in repair of defective tooth enamel affected by genetic or environmental factors. We will focus on the study of fundamental chemical and biological principles of organic matrix assembly, disassembly, degradation, and control of mineral nucleation and growth in enamel biomineralization. We will continue developing biomimetic strategies for the synthesis of enamel-like material. We hypothesize that the highly organized carbonated hydroxyapatite crystals in enamel continuously grow by means of complex protein- protein, protein-proteinase, and protein-mineral interactions that are primarily controlled by the formation of a broad range of quaternary structures of amelogenin undergoing proteolytic possessing and interacting with non-amelogenins. We further hypothesize that functional enamel-like material can be prepared in a cell-free system by using a chitosan-amelogenin as the basic structural framework and incorporating principles that we have learned and are continuing to learn from our in vitro and in vivo studies. We propose: Aim I. To investigate amelogenin-enamelin interactions at a nanoscale level in vitro and in vivo. To study the dynamics of calcium phosphate mineralization events at a nanoscale level when enamelin is combined with amelogenin, using high resolution in situ atomic force (AFM) and in situ transmission electron microscopy (TEM). Aim II. To determine the amount of protein occluded inside synthetic crystals before and after C-terminal cleavage of amelogenin as well as in enamel crystals isolated from Mmp-20 knock-out animals. To investigate the dynamics of postnucleation mineralization events at a nanoscale level during Mmp-20 proteolysis of amelogenin, using high resolution in situ AFM and TEM. Aim III. To apply chitosan-based hydrogels as mineralization matrices in the development of biomimetic strategies for re-growth of a functional enamel-like material that could be used in the clinical setting to halt incipient carious lesions. Elucidating the dynamic interactions of enamelin and Mmp-20 with amelogenin proposed in aims I- II will contribute to our understanding of the structural biology and function of the enamel extracellular matrix in vivo, and will provide a soli ground for the design and development of improved dental materials (aim III). Moreover, this study will have a significant impact on the field of biomineralization, macromolecular self-assembly, protein structure, and the understanding of pathological enamel formation.